PROJECT SUMMARY The continuing discoveries of non-coding RNAs and their critical roles in cellular and viral machinery are inspiring novel antibacterial, antitumor, antiviral, and genome-editing therapies based on disabling, manipulating, and repurposing the RNAs involved. Unfortunately, our poor biophysical understanding of `how RNAs work' hinders the development of these potentially life-saving efforts. A critical bottleneck has been the inapplicability of crystallography, NMR, cryoelectron microscopy, phylogenetic analysis, and biochemical methods to determine the partly ordered conformations of non-coding RNAs in all their functional states. To resolve this bottleneck, we are developing experimental methods and complementary computational approaches that give rich information sufficient to infer and engineer RNA secondary and tertiary structures and their heterogeneous ensembles, evaluated through community-wide blind trials, prospective compensatory mutation/rescue experiments, and global RNA design challenges. Here, we outline expansions of our research that will rigorously address four biomedically significant problems that have so far seen limited progress in molecular modeling efforts: the heterogeneity of RNA structures within their native cellular and viral contexts; modeling and design of RNA's biological interactions with proteins and other molecules, modulated by chemical modification; high-accuracy calculation of RNA folding energetics; and the automated design of dynamic 3D RNA structures for eventual medical applications. We will evaluate success through continuing blind trials, independent tests by more than a dozen expert biological and bioengineering collaborators, and through adoption of our methods and software tools by the broader research community. In the same way that specialized structural biology tools and computational design are establishing a firm understanding of protein behavior and regulation, we propose that the technologies outlined here will transform our understanding of structure in non-coding RNAs, providing a stronger basis for their biomedical activation or disruption.